**3.1 Analysis of releasable TCA content**

*Releasable TCA* values may vary depending on the cork soaking conditions: alcoholic strength of extractant, time of maceration, etc. In order to overcome these uncertainties, standardized procedures were developed. Two analytical methods proposed by OIV organization (Method OIV-MA-AS315–16 [51]) and ISO (20752:2014(E) [52]) are currently in wide use. According to these protocols, cork stoppers are macerated in an aqueous-alcoholic solution (12% vol. alcoholic strength) or white wine (10–12% vol. [51]) during 24 ± 2 h of passive soak. This time is sufficient to ensure the equilibrium for TCA extraction when it reaches a steady state [53]. Additional studies have shown that maceration time can be reduced by using active soak, for example, up to 2 hours with microwave assisted extraction (MAE) [54]. The MAE technique provides results very similar to the standard soak procedure for corks with *releasable TCA* < 25 ng/L. Once obtained, extracts are usually analyzed by GC–MS or GC-ECD in combination with headspace solid-phase microextraction (HS-SPME) [51, 52] or stir bar sorptive extraction (SBSE) [54].

The soaking of cork stoppers can be done individually or in groups. The latter approach is commonly used on an industrial scale for quality control of commercial batches of cork stoppers. Overall, comparable results have been found for group soak values and average values of individual cork soaks (R2 about 90%) [48, 53]. The size of glass containers and the volume of extractant for *releasable TCA* analysis usually depend on the number of corks. For example, group extractions of 20 and 50 corks are recommended to be done in 1 L and 2 L containers, respectively [51, 52]. There are no exact recommendations regarding the volume of extractant, but the cork stoppers should be completely immersed in the solution. It has been demonstrated that a reasonable deviation of the extractant volume does not significantly affect the TCA equilibrium and the resulting *releasable TCA* values [48]. Further studies of the adsorption/desorption process of TCA on the cork surface revealed certain limitations of the method. For example, a group soak can demonstrate an undetectable level of TCA even though some individual corks may release a certain amount of contaminant. This may occur because "clean" cork stoppers can reabsorb most of TCA from the group extract. Thus, in one study it was shown that cork stoppers are able to remove about 80% of TCA from contaminated wine after 24 h of soaking [46]. Therefore, individual soaking can be a more representative test compared with group soaking. At the same time, the results of individual soaking can also be distorted due to the reabsorption of TCA by "clean" parts of the same cork.

*State-of-the-Art Knowledge about 2,4,6-Trichloroanisole (TCA) and Strategies to Avoid… DOI: http://dx.doi.org/10.5772/intechopen.103709*

Despite the described adsorption/desorption effects, the values of *releasable TCA* analysis for individual stoppers correlated quite well with the TCA content in wines bottled with the same corks [48]. Thus, it was found that 14 months after bottling, on average, the concentration of TCA in wines was about half the corresponding *releasable TCA* values. The lower TCA content in real conditions can be due to the fact that the wine contacts only a limited surface of the cork in the bottle, while during the *releasable TCA* analysis, the entire cork is immersed in the extractant.

For the analysis of cork extracts, the same GC methods are used as for the analysis of wine [55, 56]. Therefore, in addition to TCA, other haloanisoles (TeCA, PCA, TBA, etc.) and halophenols can also be quantified. For a more accurate determination of the latter (TCP, TeCP and PCP), preliminary derivatization of extracts (acetylation) can be carried out [57]. Finally, in addition to GC methods, a bioanalytical technique for the analysis of wine and cork extracts (Bioelectric Recognition Assay (BERA)) was studied [58]. This technique is based on a biosensor containing membrane-engineered cells with inserted TCA-specific antibodies. Therefore, it is limited only to the TCA determination and operates in the range of about 1–12 ng/L. On the other hand, BERA is a relatively fast analysis, requiring only 3–5 min, and can be considered as a promising express method.

#### **3.2 Analysis of total TCA content**

The key concept of this method is the maximum extraction (recovery) of hydrophobic haloanisoles from the cork matrix. This can be achieved by selecting an effective solvent and grinding the cork to obtain a large surface in contact with the extractant. Corks can be ground in a granulating mill with a stainless steel bowl [59] or in a regular coffee grinder [46]. It is recommended to pre-freeze corks to facilitate the grinding process and prevent the loss of volatile organic compounds due to evaporation. Freezing can be done by immersing a cork stopper in liquid nitrogen [60, 61]. To increase the repeatability of the analysis, it is recommended to make the fraction of ground cork less than 3 mm [35] or even homogenize it by passing it through a sieve, e.g., 1 mm in diameter [61, 62]. At the same time, the analysis of pieces around 5 x 5 mm also demonstrated good recoveries and repeatability [63].

Among the tested solvents, hexane and pentane showed high extractive properties with respect to hydrophobic haloanisoles and are now widely used [2, 63, 64]. According to the OIV protocol, an ethyl ether/hexane mixture (50/50; v/v) is recommended [35]. Alcoholic solutions with an ethanol concentration of more than 50% (vol.) showed lower but still good results. In particular, a solution with 75% (vol.) of ethanol can be recommended in certain situations, for example, in the case of a subsequent SBSE analysis technique [59]. Methanol in combination with some extraction methods is also a good candidate for analysis [62]. Other solvent options have also been described, but they are not widely used or are specified for certain extraction methods: pentane/ethyl acetate [4], pentane/diethyl ether for pressurized liquid extraction (PLE) method [60], etc.

With regard to extraction techniques, there are several approaches that include conventional soak, Soxhlet extraction, and various advanced methods. Conventional soak of ground cork is usually performed in closed glass vessels, and variations are related to the selection of solvent, extraction time, application of mechanical agitation, etc. Generally, the method is effective, but time-consuming: typically maceration takes 24 hours without mechanical agitation [37, 46, 63, 65]. Maceration time can be significantly reduced by using agitation in a rotary mixer

[64] or vortex [35], by sonication in an ultrasonic bath (15–30 min) [59, 64] or immersing an ultrasonic processor inside the cork/solvent mixture for 1–2 min [66, 67]. Conventional soak is an effective method with the possibility to achieve TCA recoveries of more than 90% [50].

Soxhlet apparatus provides continuous circulation of a boiling extractant through a ground cork. Extraction time usually varies between 7 and 24 hours [33, 62, 68], making this method not time-efficient. Nowadays, Soxhlet extraction is less often used as a routine technique, but remains a reliable reference method due to its high TCA recovery (up to 99%), repeatability, reproducibility, and small deviation between replicates [62, 64].

Both conventional soak and Soxhlet extraction result in a relatively large amount of extract, which must be concentrated prior to injection for GC analysis. Therefore, the improvement of extraction methods was aimed not only at optimizing the time, but also at reducing the volume of solvent used. It has been proposed to utilize the following special extraction techniques for haloanisoles: microwaveassisted extraction—MAE [62], supercritical fluid extraction—SFE [69], pressurized liquid extraction—PLE [60], pressurized fluid extraction—PFE [70], etc. All of these advanced extraction methods demonstrated excellent efficiency (high recoveries and good reproducibility), but they require specific equipment.

The next steps in the development of cork analysis are organic solvent-free methods, which involve heating ground cork with or without water. As a result, TCA and other haloanisoles are vaporized and then analyzed, for example, using HS-SPME [61, 71]. These methods of direct analysis do not require special sample preparations, but are carried out with a smaller amount of analyzed cork, e.g., 200 mg or less. A similar approach was also proposed for the analysis of entire natural corks and is discussed in Section 4.2.2.

Finally, the determination of *total TCA* and other haloanisoles and halophenols can be performed not only for cork stoppers, but also for various objects present in cellars: wooden pallets [33], oak barrel sawdust [72, 73], wooden chips [35], wooden staves [74], and other cellar materials [39]. All of these materials should be preliminary ground, as it is required for corks [35].

#### **4. Strategies to avoid TCA presence in wine**

It is more practical to prevent TCA contamination of wines during their production, bottling, and storage process than to remove the *cork taint* later. Strategies for avoiding haloanisoles pollution of the winery environment, equipment, enological products are well described by Jung and Schaefer [27] and are partially mentioned in *Section 2.2* of this chapter. The current section will discuss how to reduce/eliminate TCA contamination in cork stoppers. Being among the most unpleasant and most frequent wine defects, the *cork taint* problem has triggered numerous research projects led by cork industry players over the last 30 years to remedy this situation.

One group of the early methods was aimed at sterilization of cork material (to eliminate microorganisms producing TCA) and decontamination. The corresponding technologies involved exposure of cork material to microwave radiation [75]; treatment of cork with alkaline solutions [76, 77], etc. Other methods were focused on the elimination of chlorophenols (TCA precursors) from the cork material, such as treatment of cork with a phenol oxidizing enzyme [78] or application of *Chrysonilia sitophila* fungi, which are able to degrade TCP without formation of TCA and inhibit growth of TCA-producing fungi [79].

The use of physical barriers on a cork stopper to prevent it from making contact with bottled wine is another strategy that has been tested. For example, a silicon joint *State-of-the-Art Knowledge about 2,4,6-Trichloroanisole (TCA) and Strategies to Avoid… DOI: http://dx.doi.org/10.5772/intechopen.103709*

on champagne stoppers was studied to prevent the migration of TCA into wine [80]. Another study investigated a nanostructured carbon-based film on the cork surface as a barrier against dust and impurities that may penetrate and pollute the cork mass [81].

Most of the approaches listed above demonstrated only limited effectiveness in diminishing TCA in cork stoppers and its appearance in bottled wine. Therefore, there are currently two main strategies for dealing with contaminated corks:


More details on these strategies, their limitations, and associated difficulties are presented in the following subsections.

#### **4.1 Technical methods to reduce/eliminate TCA presence in cork stoppers**

Various products and techniques were proposed for cleaning and eliminating TCA from contaminated corks: for example, treatment with an aqueous suspension of activated charcoal [82] or a mixture of water and organic solvents (including ethanol) combined with a heating phase obtained with electromagnetic energy at hyper frequencies [83], etc. Not all tested cork cleaning methods have shown high efficiency, reasonable installation costs, processing and energy consumption, as well as safety requirements. In addition, some processes can have secondary effects that cause significant changes in the physical and chemical composition of corks, leading to the alteration of their mechanical or sensory properties. As a result, there are a limited number of cork cleaning technologies that have proven their practical applicability and suitable for use on an industrial scale. Among these approaches are treatment with steam, thermal desorption by vacuum, and treatment with supercritical CO2.

#### *4.1.1 Treatment with steam*

It is known that the concentration of TCA in cork can be diminished by simple aeration, which can be accelerated by higher temperature and humidity [37, 84]. Therefore, steam distillation technique was proposed to remove volatile substances, including TCA.

Steam extraction technologies are used nowadays by different cork manufacturers and demonstrate good results (**Figure 7**). For example, the first industrial steam cleaning process ROSA® of Amorim Cork provided the removal of about 80% of TCA from cork granules [86], which are then used to produce agglomerated cork stoppers. Subsequent optimization of the process led to a reduction in the TCA content to almost "zero" level (i.e., below the limit of quantification (LOQ )) for cork granules, which possessed the initial *releasable TCA* levels less than 6 ng/L. The next development step allows treating entire natural cork stoppers (ROSA Evolution®), reducing their *releasable TCA* levels by 80–85%.

Other companies that use steam to clean natural corks and granules also have their own particularities in the process (Innocork® and Vapex®, by Cork Supply; Neotech® and Sara Advanced®, by M.A.Silva; Revtech and others). For example, utilization of an ethanol-water vapor mixture to treat corks (Innocork®). The process can take place under 60°C allowing reduction of the TCA content up to 80% [87]. Higher temperatures above 70–80°C are not recommended, because they led to irreversible distortions of the stoppers after cooling [87]. Atmospheric pressure


**Figure 7.**

*Steam extraction technology (ROSA®) for TCA extraction from cork granules [85].*

is suitable for these cleaning technologies as it provides good extraction results at a considerable cost reduction (no special low-pressure equipment is required). At the same time, a higher or lower pressure (0.2–0.8 bars) or a variation of pressure in the cleaning system can be applied to increase the efficiency of TCA removal. For example, Belighit with colleagues (2010) proposed cycles of pressurization with water vapor followed by periods of vacuum to enhance the cork cleaning [88].
